Nitrate respiration in Pseudomonas stutzeri A15 and its involvement in rice and wheat root colonization

Unlike most bacteria, the nitrogen-fixing rice-associated Pseudomonas stutzeri A15 disposes of three different nitrate reductases that enable conversion of nitrate to nitrite through three physiologically distinct processes, called nitrate assimilation, nitrate respiration and nitrate dissimilation. To study the role of nitrate respiration in rhizosphere fitness, a Pseudomonas stutzeri narG mutant was constructed and characterized by assessing its growth characteristics and whole-cell nitrate reductase activity in different oxygen tensions. Unexpectedly, the Pseudomonas stutzeri A15 narG mutant appeared to be a better root colonizer, outcompeting the wild type strain in a wheat and rice hydroponic system.     

Complete genome sequence of the nitrogen-fixing and rhizosphere-associated bacterium Pseudomonas stutzeri strain DSM4166

We present here the analysis of the whole-genome sequence of Pseudomonas stutzeri strain DSM4166, a diazotrophic isolate from the rhizosphere of a Sorghum nutans cultivar. To our knowledge, this is the second genome to be sequenced for P. stutzeri. The availability and analysis of the genome provide insight into the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in interactions with host plants.     

Characterization of Esterase A, a Pseudomonas stutzeri A15 Autotransporter

Autotransporters are a widespread family of proteins, generally known as virulence factors produced by Gram-negative bacteria. In this study, the esterase A (EstA) autotransporter of the rice root-colonizing beneficial bacterium Pseudomonas stutzeri A15 was characterized. A multiple sequence alignment identified EstA as belonging to clade II of the GDSL esterase family. Autologous overexpression allowed the investigation of several features of both autotransporter proteins and GDSL esterases. First, the correctly folded autotransporter was shown to be present in the membrane fraction. Unexpectedly, after separation of the membrane fraction, EstA was detected in the N-laurylsarcosine soluble fraction. However, evidence is presented for the surface exposure of EstA based on fluorescent labeling with EstA specific antibodies. Another remarkable feature is the occurrence of a C-terminal leucine residue instead of the canonical phenylalanine or tryptophan residue. Replacement of this residue with a phenylalanine residue reduced the stability of the β-barrel. Regarding the esterase passenger domain, we show the importance of the catalytic triad residues, with the serine and histidine residues being more critical than the aspartate residue. Furthermore, the growth of an estA-negative mutant was not impaired and cell mobility was not disabled compared to the wild type. No specific phenotype was detected for an estA-negative mutant. Overall, P. stutzeri A15 EstA is a new candidate for the surface display of proteins in environmentally relevant biotechnological applications.     

Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001

A strain YZN-001 was isolated from swine manure effluent and was identified as Pseudomonas stutzeri. It can utilise not only nitrate and nitrite, but also ammonium. The strain had the capability to fully remove as much as 275.08 mg L⁻¹ NO₃⁻–N and 171.40 mg L⁻¹ NO₂⁻–N under aerobic conditions. Furthermore, At 30 °C, the utilization of ammonium is approximately 95% by 18 h with a similar level removed by 72 h and 2 weeks at 10 and 4 °C, respectively. Triplicate sets of tightly sealed serum bottles were used to test the heterotrophic nitrifying ability of P. stutzeri YZN-001. The results showing that 39% of removed NH₄⁺–N was completely oxidised to nitrogen gas by 18 h. Indicating that the strain has heterotrophic nitrification and aerobic denitrification abilities, with the notable ability to remove ammonium at low temperatures, demonstrating a potential using the strain for future application in waste water treatment.     

Construction of a stable expression vector for Leifsonia xyli subsp. cynodontis and its application in studying the effect of the bacterium as an endophytic bacterium in rice

To study the possibility of utilizing genetically engineered Leifsonia xyli subsp. cynodontis (Lxc) as an endophytic bacterium in rice, we constructed an Escherichia coli-Lxc shuttle vector, pLGUS, containing a beta-glucuronidase reporter gene, which was stable both in vitro and in vivo. Lxc grows and expresses the beta-glucuronidase reporter gene in all parts of rice, except for seed. A 2-year field study using three rice varieties from China showed that Lxc inoculation did not have a negative effect on the growth and yield of any of these varieties. Therefore, Lxc has the potential to be used as a benign endophyte for the expression of foreign genes in rice.     

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes

Aims: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Methods and Results: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but β‐glucuronidase (GUS)‐stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5–plant interaction. PAL5 could be isolated from the root surface (10⁸ CFU g^?1) and from surface‐disinfected root and stem tissues (10⁴ CFU g^?1) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. Conclusion: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. Significance and Impact of the Study: These tools are of use to: (i) study PAL5 mutants affected in bacteria–plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.     

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

Cytochrome systems in cells of a denitrifying bacterium, Pseudomonasstutzeri (VAN NIEL strain), grown under different atmosphericconditions were compared with reference to the effects of nitrateand nitrite on cytochrome synthesis. When a culture was sufficiently aerated (aerobic conditions),synthesis of all cytochrome components was repressed, regardlessof the presence or absence of nitrate and nitrite. When aeratedmoderately (semi-aerobic conditions), both soluble and paniculatecytochromes c-552 and cytochrome b-558 contents markedly increasedeven in the absence of nitrate and nitrite. Under anaerobic or semi-aerobic conditions, nitrite inducedcytochrome a₂–c synthesis. This inductive effect of nitritewas counteracted by nitrate. Nitrate also repressed particulatecytochrome c-552 synthesis to some extent but nitrite did not. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan (Received June 24, 1969; )     

A species-specific probe and a PCR assay for the marine bacterium,Pseudomonas stutzeri strain Zobell

The cloning, sequencing, and analysis of a Pseudomonas stutzeri Zobell 23S rRNA gene is described. Three variable regions were identified, and oligonucleotides homologous to portions of these regions were synthesized. The oligonucleotides were used as probes to screen DNA from various cultured bacteria to identify a species-specific probe. All probes were found to hybridize strongly with P. stutzeri Zobell DNA under stringent conditions and did not hybridize with other Pseudomonas species. One probe showed slight cross-reactivity with DNA from four other bacteria under the hybridization conditions used. Finally, PCR conditions were optimized for detection of P. stutzeri Zobell in mixed culture with a detection limit of 400 cells. The assay detected P. stutzeri Zobell rDNA in coastal seawater samples sampled over a 20-month period. In the future, these probes could be used to quantify the 23S rRNA and rDNA from P. stutzeri Zobell in mixed culture and in environmental samples.     

Detection and characterization of natural transformation in the marine bacterium Pseudomonas stutzeri strain ZoBell

Soil isolates of Pseudomonas stutzeri have been shown previously to acquire genes by natural transformation. In this study a marine isolate, Pseudomonas stutzeri strain ZoBell, formerly Pseudomonas perfectomarina, was also shown to transform naturally. Transformation was detected by the Juni plate method and frequencies of transformation were determined by filter transformation procedures. Maximum frequencies of transformation were detected for three independent antibiotic resistance loci. Transformation frequencies were on the order of 4×10^-5 transformants per recipient, a frequency over 100 times that of spontancous antibiotic resistance. Transfer of antibiotic resistance was inhibited by DNase I digestion. Marine isolates achieved maximum competence 14 h after transfer of exponential cultures to filters on solid media, although lower levels of competence were detected immediately following filter immobilization. Like soil isolates, P. stutzeri strain ZoBell is capable of cell contact transformation, but unlike soil isolates where transformation frequencies are greater for cell contact transformation as compared to transformation with purified DNA, the maximum frequency of transformation achieved by cell contact in the marine strain was approximately 10-fold less than transformation frequencies with purified DNA. These studies establish the first marine model for the study of natural transformation.This paper is dedicated to John L. Ingraham, Professor Emeritus of Microbiology at the University of California, Davis. Professor Ingraham was the first person to recognize natural transformation in Pseudomonas stutzeri and has continued to contribute to our understanding of the process over the past eight years. This understanding of the genetics of P. stutzeri is only one of the many areas of microbiology to which Professor Ingraham has contributed in his exceptional career     

Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67

A beta-glucoronidase (GUS)-marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedling cvs. IR42 and IR72. Internal populations peaked at over 10(6) log CFU per gram of fresh weight by 5 to 7 days after inoculation (DAI) but declined to 10(3) to 10(4) log CFU per gram of fresh weight by 28 DAI. GUS staining was most intense on coleoptiles, lateral roots, and at the junctions of some of the main and lateral roots. Bacteria entered the roots via cracks at the points of lateral root emergence, with cv. IR72 appearing to be more aggressively infected than cv. IR42. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. Xylem vessels in leaves and stems were extensively colonized at 2 DAI but, in later harvests (7 and 13 DAI), a host defense reaction was often observed. Dense colonies of H. seropedicae with some bacteria expressing nitrogenase Fe-protein were seen within leaf and stem epidermal cells, intercellular spaces, and substomatal cavities up until 28 DAI. Epiphytic bacteria were also seen. Both varieties showed nitrogenase activity but only with added C, and the dry weights of the inoculated plants were significantly increased. Only cv. IR42 showed a significant (approximately 30%) increase in N content above that of the uninoculated controls, and it also incorporated a significant amount of 15N2.     

Transfer and expression of Klebsiella nif genes in Alcaligenes faecalis, a nitrogen-fixing bacterium associated with rice root

Plasmid pRD1 carrying Klebsiella nif genes was found to be transferable by conjugation from Escherichia coli JC5466 (pRD1) to Alcaligenes faecalis A-15 at a frequency of 5 X 10(-4). Nitrogenase activity of four A-15 (pRD1) strains tested was found to be higher than that of their parent A-15, as determined by the acetylene reduction assay. A-15-1 was a Nif- mutant derived from A-15. After mating with JC5466 (pRD1), the nitrogenase activity was restored. PRD1 was stable in A. faecalis and could be transferred to E. coli JC5466-1 by conjugation. The fact that Klebsiella nif genes carried in pRD1 can be expressed in A. faecalis makes it possible to use pRD1 as a tool for genetic analysis and genetic engineering of the nitrogen fixation system in A. faecalis.     

Reduction of Selenate and Selenite to Elemental Selenium by a Pseudomonas stutzeri Isolate

A Pseudomonas stutzeri isolate rapidly reduced both selenite and selenate ions to elemental selenium at initial concentrations of both anions of up to 48.1 mM. Optimal selenium reduction occurred under aerobic conditions between pH 7.0 and 9.0 and at temperatures of 25 to 35°C. Reduction of both selenite and selenate was unaffected by a number of anions except for sulfite, chromate, and tungstate ions, which inhibited both growth and reduction.     

Maximizing growth and productivity of onion (Allium cepa L.) by Spirulina platensis extract and nitrogen-fixing endophyte Pseudomonas stutzeri

Archives of Microbiology - The study focuses on the impact of foliar spraying cyanobacterium Spirulina platensis extract and the inoculation with the endophyte N2-fixing Pseudomonas stutzeri, and...     

The biochemical pathway for the breakdown of N4-ethyl-L-asparagine in the bacterium Pseudomonas stutzeri.

N4-Ethyl-L-[u-14C]asparagine and L-[U-14C]aspartate give identical metabolites, mainly intermediates of the tricarboxylic acid cycle and related amino acids, in whole cells of Pseudomonas stutzeri. The labelled asparagine derivative is converted into [14C]-aspartate by cell-free extracts, and this reaction, which has an optimum pH of 8.8 +/- 0.2, is neither inhibited by unlabelled asparagine nor enhanced by unlabelled 2-oxoglutarate. No labelled keto acid corresponding to N4-ethylasparagine was detected in either whole cells or cell-free extracts. Thus N4-ethyl-L-asparagine, like asparagine, must be broken down by hydrolysis, at least in this bacterium.     

Interference of quorum sensing and virulence of the rice pathogen Burkholderia glumae by an engineered endophytic bacterium

Many bacterial species are known to thrive within plants. Among these bacteria, a group referred to as endophytes provide beneficial effects to the host plants by the promotion of plant growth and the suppression of plant pathogens. Among 44 putative endophytic isolates isolated from surface-sterilized rice roots, Burkholderia sp. KJ006 was selected for further study because of a lack of pathogenicity to rice, a broad spectrum of antifungal properties, and the presence of the nifH gene, which is an indicator for nitrogen fixation. In an attempt to control Burkholderia glumae, a casual pathogen of seedling rot and grain rot of rice, an N-acyl-homoserine lactonase (aiiA) gene from Bacillus thuringiensis was introduced into Burkholderia sp. KJ006 given that the major virulence factor of Burkholderia glumae is controlled in a population-dependent manner (quorum sensing). The engineered strain KJ006 (pKPE-aiiA) inhibited production of quorum-sensing signals by Burkholderia glumae in vitro and reduced the disease incidence of rice seedling rot caused by Burkholderia glumae in situ. Our results indicate the possibility that a bacterial endophyte transformed with the aiiA gene can be used as a novel biological control agent against pathogenic Burkholderia glumae that are known to occupy the same ecological niche.     

Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61

The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334 amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K _m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61. Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998     

Probing the applicability of autotransporter based surface display with the EstA autotransporter of Pseudomonas stutzeri A15

Buffering to achieve pH control is crucial for successful trichloroethene (TCE) anaerobic bioremediation. Bicarbonate (HCO3−) is the natural buffer in groundwater and the buffer of choice in the laboratory and at contaminated sites undergoing biological treatment with organohalide respiring microorganisms. However, HCO3− also serves as the electron acceptor for hydrogenotrophic methanogens and hydrogenotrophic homoacetogens, two microbial groups competing with organohalide respirers for hydrogen (H2). We studied the effect of HCO3− as a buffering agent and the effect of HCO3−-consuming reactions in a range of concentrations (2.5-30 mM) with an initial pH of 7.5 in H2-fed TCE reductively dechlorinating communities containing Dehalococcoides, hydrogenotrophic methanogens, and hydrogenotrophic homoacetogens. Rate differences in TCE dechlorination were observed as a result of added varying HCO3− concentrations due to H2-fed electrons channeled towards methanogenesis and homoacetogenesis and pH increases (up to 8.7) from biological HCO3− consumption. Significantly faster dechlorination rates were noted at all HCO3− concentrations tested when the pH buffering was improved by providing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as an additional buffer. Electron balances and quantitative PCR revealed that methanogenesis was the main electron sink when the initial HCO3− concentrations were 2.5 and 5 mM, while homoacetogenesis was the dominant process and sink when 10 and 30 mM HCO3− were provided initially. Our study reveals that HCO3− is an important variable for bioremediation of chloroethenes as it has a prominent role as an electron acceptor for methanogenesis and homoacetogenesis. It also illustrates the changes in rates and extent of reductive dechlorination resulting from the combined effect of electron donor competition stimulated by HCO3− and the changes in pH exerted by methanogens and homoacetogens.